Calcium transport across the intestine requires the entry of calcium into the intestinal epithelial cell across the brush border membrane (BBM), passage through the cell, and removal at the basolateral membrane, 1,25(OH)2D regulates calcium transport at each of these steps but the molecular mechanisms are poorly understood. At the BBM calcium transport is increased by 1,25(OH)2D presumably by changes in the function of an as yet unidentified carrier or channel. Associated with the increases in calcium transport across the BBM is an increase in calmodulin (CaM) binding to a 102,000 mol wt protein in the BBM; CaM antagonists block 1,25(OH)2D stimulated calcium transport across the BBM and block the binding of CaM to this protein. Neither the increased calcium transport nor the increased CaM binding to this protein following 1,25(OH)2D are blocked by bycloheximide despite marked inhibition of protein synthesis. Our hypothesis is that this 102,000 mol wt protein is the calcium channel or a modulator of the calcium channel in the BBM and is central to 1,25(OH)2D regulation of calcium transport across the BBM. We plan to purify this protein by a combination of membrane extraction, salt precipitation, HPLC, and SDS polyacrylamide gel electrophoresis. The purified protein will be evaluated for its capacity to act as a calcium channel or a regulator of such a channel by reconstitution experiments with liposomes. Comparison of the protein isolated from vitamin-D deficient and vitamin-D replete chicks will be made with respect to CaM binding and calcium inophore activity. Changes in phosphorylation and glycosylation of this protein after 1,25(OH)2D administration will be sought. The protein will be used as antigen to develop a polyclonal antiserum for immunolocalization studies, immunoassays, Western blots, and affinity chromatography. From the purified protein partial amino acid sequences will be determined to guide the synthesis of oligonucleotide probes which will be used to select the appropriate clone(s) from a cDNA library prepared from chick intestinal mRNA. After identifying and sequencing the clones spanning the reading frame for the protein, the primary amino acid structure will be deduced, and computer searches made to identify homologous sequences in other proteins. Regions of particular interest such as the CaM binding domain and transmembrane domains will be sought and compared to other CaM binding proteins and ion channels. From these studies a clearer molecular understanding of calcium transport across the BBM should emerge.